1. Field of the Invention
The present invention relates to a technique for cell fusion of plants, more particularly to a method for promoting fusion of plant protoplasts. The fused cells obtained by the cell fusion technique have new characteristics. Large-scale culture of the cells, for example, can provide a useful secondary metabolite product, or the fused cells differentiate to a useful new plant.
2. Description of the Prior Art
Rapid progress in tissue culture techniques has led to techniques for breeding new hybrid plants by artificial cell fusion. Breeding of hybrids of distantly related plants, hitherto thought impossible, is now feasible.
In cell fusion, the cell wall of the plant cells is removed by enzymes or a mechanical means, and the protoplasts are mixed to prepare fused cells. Simple mixing of the protoplasts, however, provides only a very low fusion rate. Therefore, various methods have been proposed for promoting the fusion of protoplasts.
The most preferable of these methods is to treat the protoplasts by a fusion agent prepared by a combination of polyethylene glycol and an alkaline aqueous solution of calcium chloride. It is possible to further improve the fusion by (1) raising the temperature, (2) using a decomposing agent for membrane protein of cell membrane, or (3) carrying out centrifugation.
While such methods may improve the fusion rate somewhat, the rate attained is still not satisfactory. Moreover, the methods cannot effect the cell fusion in some kind of plants.
Now, in the prior art cell fusion method, after the protoplasts are treated by the fusion agent, the fusing agent is removed by repeated washing with an aqueous solution. Then, the washed protoplasts are placed in the aqueous solution for storage.
To store the protoplasts in a stable form, it is necessary to raise the osmotic pressure of the aqueous solution carrying the protoplasts.
Toward this end use is made of an aqueous solution containing, for example, a sugar such as sucrose or glucose, a sugar alcohol such as mannitol or sorbitol, or an inorganic salt such as calcium chloride or potassium chloride as an osmotic pressure control agent. The osmotic pressure of the aqueous solution is preferably adjusted to between 12 kg/cm.sup.2 and 30 kg/cm.sup.2, more preferably to between 12 kg/cm.sup.2 and 20 kg/cm.sup.2.